In the field of life science, it is very important to analyze various phenomena that occur in cells, e.g., to monitor changes of intracellular calcium ion concentration, phosphorylation of intracellular proteins, distributions of ATP, which is an energy, or transcription activities of the genes. In order to analyze them, various molecular probes have been developed and applied for imaging. In particular, various fluorescent proteins are used as tools for cell imaging. Fluorescent protein fluoresces without a cofactor immediately after being expressed in a cell. The fluorescent protein is utilized as a monitor protein for localization of the protein in a cell, using fluorescence as an indicator. However, quantification is difficult because of the requirement of an excitation light and uneven fluorescence efficiency, and the cells are impaired because of exposure to the excitation light. Thus, fluorescent protein is not suitable for long-term observation.
The measurement of the transcription activity using a reporter gene is the tool used to analyze various intracellular molecular mechanisms, e.g., the analysis of the activation of intracellular signal transduction or the analysis of receptor-ligand interaction by measuring an expressed amount of the reporter gene linked to a certain promoter, in addition to the analysis of a gene expression regulatory sequence such as a promoter, an enhancer and a silencer or a transcription factor bound thereto. This technique is used as a large-scale screening tool in drug discovery and toxicity evaluation of chemicals.
The reporters used here include many enzymes such as chloramphenicol acetyl transferase (CAT), β-galactosidase, and green fluorescent protein (GFP). A system using the bioluminescence of firefly luciferase is widely used currently because it is highly sensitive and simpler to be assayed than other reporter enzyme. GFP does not require a substrate and can be easily detected by irradiating the excitation light, but is not suitable for quantification. Since the excitation light is irradiated, the cells are greatly damaged. Thus, GFP is not suitable for long-term, continuous monitoring purposes.
Firefly luciferase is luciferase derived from luminescent beetles, and cDNA thereof has been isolated from the fireflies belonging to genera Photinus, Photuris and Luciola. In particular, the gene derived from Photinus pyralis has been studied in detail over the years. Luciferases derived from beetles including the firefly act on a poly-heterocyclic organic acid, D-(−)-2-(6′-hydroxy-2′-benzothiazolyl-Δ2-thiazoline-4-carboxylic acid (hereinafter represented as luciferin) as a substrate, and catalyze a reaction of ATP and luciferin in the presence of Mg ion to form luciferyl adenylate, which is bound to oxygen to generate oxyluciferin in an excited state. Luminescence is emitted when this oxyluciferin relaxes to a ground state.
Firefly luciferase is used as the reporter gene for the evaluation of effects of exogenous factors on the cells, propagation of the intracellular signal transduction or expression of individual proteins. A system is included in which the amount of luciferase synthesized intracellularly is measured to evaluate transcriptional activity by linking a transcriptional regulatory region to a firefly luciferase gene, introducing the gene construct into cells, treating the cultured cells transfected with the reporter gene with a drug for a certain time period, and subsequently collecting the cells and adding a luminescent substrate. The system has excellent quantitative properties because transcriptional activity is evaluated with the luminescence amount of luciferase, and products related to this system have been developed and made commercially available from many companies.
As the imaging using firefly luciferase, for example, the change of an ATP in the cells has been successfully visualized by measuring the ATP amount using firefly luciferase, in which the change of the intercellular calcium concentration being visualized using a photoprotein, Aequorin (Non-Patent Literature 1). Another example discloses an intermolecular force between the proteins successfully visualized by a firefly luciferase split assay (Non-Patent Literature 2). Although luciferase imaging is not as suitable as fluorescent proteins for analysis at molecular level and for microscopic imaging inside a cell, it enables the obtainment of cellular information that cannot be measured using fluorescent protein in the analysis of the phenomena that occur in the cells at an organelle level, and particularly in long-term measurement. Firefly luciferase imaging is an effective method for the evaluation and screening of pharmaceuticals.
However, there are few examples of firefly luciferase imaging. This is because the stability of luciferase in mammalian cells is lower, the protein lifespan is shorter compared with the fluorescent protein, and the transcription efficiency is low; firefly luciferase is thus not suitable for practical use, as image analyzers of the cells correspond to the fluorescence, and no imaging system for efficiently measuring the luminescence is available. This is particularly because the luminescence intensity of firefly luciferase in living cells is low, thus hindering the easy obtainment of luminescence signals.
Among firefly luciferases, the enzyme used most frequently for imaging and the like is luciferase derived from fireflies produced in North America (Photinus pyralis). It has been reported recently that the mutants having improved thermal stability and their half-lives prolonged by about 2 to 25 times in vitro emits enhanced luminescence signal in the cells and are suitable for cell imaging (Non-Patent Literature 3). However, one shortcoming of firefly luciferase is that the luminescent color is changed in conjunction with the intracellular pH value, and thus is not suitable for analyzing multiple gene expressions based on the diversity of luminescent colors (Yoshihiro Ohmiya, Yoshihiro Nakajima, Multiple Gene Transcription Activity Measurement System; Patent Document 1).
Luciferase expressed in an animal or a cell in which a beetle-derived luciferase gene has been introduced can be detected with its luminescence by administering luciferin into the animal, or by adding luciferin to the cell culture and permeating it into the cell to perform a luciferase-luciferin reaction.
However, in many cases, luciferase expressed in the cell is detected by lysing the cell with a reagent containing a surfactant, mixing the luciferase-containing cell lysate with the luminescent substrate reagent and measuring the luminescence of luciferin. This method is complicated because the cell is lysed and luciferase further reacted with the luminescent reagent, compared with the method of detection adding luciferin to the cell culture medium. Once the cell is lysed, further phenomenon in the cell cannot be observed. Meanwhile, one merit of the method above, in which the cell is not lysed, is that the intracellular phenomenon can be continuously observed by prolonging a culture time period as needed. Despite its drawbacks, the cell-lysing detection method is currently mainstream. The major reasons for this are that the firefly luciferase reporter does not react sufficiently with luciferin in the living cells, the signals are weak and the sensitivity is low. These shortcomings become remarkable when a promoter having weak transcription activity is analyzed or a transient assay of the reporter gene is performed using cells having a low gene introduction efficiency. Additionally, when multiple samples are measured simultaneously, the sample amount is reduced and measurement is difficult in a detection system with low sensitivity. Thus, a reporter assay method in which the transcriptional activity can be monitored efficiently in living cells has been required.
Patent Document 1: WO2004/99421    Non-Patent Literature 1: Sala-Newby G B et al.: Imaging bioluminescent indicators shows Ca2+ and ATP permeability thresholds in live cells attacked by complement. Immunology. 1998 April; 93 (4):601-9    Non-Patent Literature 2: Ozawa T. et al.: Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal. Chem., 2001 Jun. 1; 73 (11):2516-21    Non-Patent Literature 3: Baggett B. et al., Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence. Mol. Imaging, 2004 October; 3 (4):324-32.
It is important when developing or evaluating a drug, or when evaluating the toxicity of chemicals, to evaluate the effects of a exogenous factor on a living body. Evaluation using organism individuals such as mice and evaluation using tissues and cell populations have advanced, and further, intercellular and intracellular information changes are currently examined at single cell level to evaluate the exogenous factor. Thus, a molecular probe for evaluating the intercellular and intracellular information change becomes important for evaluating the exogenous factor. The fluorescent protein is suitable as an intracellular imaging probe for short-term measurement, but is not suitable for long-term analysis. Luciferase is suitable for long-term measurement, but has not been established as an imaging tool.
To establish luciferase as an intracellular and intercellular imaging tools, high stability and a relatively long protein lifespan of luciferase in the mammalian cell are desired. It is also desired that luciferase fused with various tag protein domains have high luminescence intensity. The sufficient luminescence intensity and stability were not found in luciferase derived from the fireflies produced in North America and Japan conventionally used for measuring the transcriptional activity in the mammalian cells. In particular, stable measurements are difficult in luciferases derived from fireflies produced in North America and Japan because the luminescence spectra changes depending on environmental pH values.